Systemic bacterial infection caused by Gram-negative bacteria represents a serious health problem resulting in over 100,000 deaths per year in the United States. At least 1% of all patients admitted to hospitals either have or acquire, during their stay, Gram-negative septicemia. Once a patient enters into septicemic shock the death rate is about 50%. It is therefore critical that these infections be detected at the preshock stage.
Classically bacterial infections are detected by culturing a sample of a body fluid for 2-14 days, measuring bacterial growth, and identifying the organisms.
It is known that bacteria release endotoxins during lysis. Endotoxins are lipopolysaccharides associated with various amounts of protein and lipid, the biological activity residing primarily with the lipopolysaccharides.
When admitted to the bloodstream of animals endotoxins initiate a complex chain of biochemical reactions which result in an elevation in body temperature (fever) and as a result these substances have been referred to as pyrogens.
In fact, a commonly used test for detecting endotoxins in intravenous solutions and other parenterally administered pharmaceutical preparations is the so-called rabbit fever test. In this test, rabbits are injected with a portion of the parenteral preparation to be tested and their temperature is monitored, an elevated temperature being a positive test for the presence of endotoxins (pyrogens).
Endotoxins can also be detected in picogram/ml quantities by the Limulus Amoebocyte Lysate method, described in New England Journal of Medicine, Vol. 289, No. 18, 931-934 (1973) and U.S. Pat. No. 4,107,007, Aug. 15, 1978. Limulus amoebocyte lysate is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. The endotoxins activate an enzyme in the Limulus amoebocyte lysate which then reacts with low molecular weight clottable proteins to form a gel. Typically, 0.1 ml solution of a freeze-dried Limulus lysate preparation sold commercially under the trademark Pyrotell.RTM. by Associates of Cape Cod, Inc. is mixed with 0.1 ml of a test sample, then allowed to incubate undisturbed for one hour at 37.degree. C. A positive test is indicated by the formation of a gel which does not collapse upon 180 degree inversion of the test tube.
Despite the existence of methods for detecting endotoxins at extremely low concentrations and the critical need to detect endotoxins in body fluids, there presently are no reliable methods for detecting endotoxin levels in body fluids such as blood, plasma, and serum. There are a number of difficulties residing in measuring endotoxins in body fluid.
The biological activities of endotoxins in body fluids are to varying degrees masked by the association of the endotoxin with protein and lipid.
For example, Journal of Bacteriology, 92, No. 4, 892-892, (1966) describes alteration and restoration of endotoxin activity after complexing with plasma protein. Also see Annals of the New York Academy of Sciences, Vol. 133, 629-643 (1966) for a discussion of the physical aspects of reversible inactivation of endotoxins.
It is therefore recognized that chemical dissociation of endotoxin complexes in biological fluids will unmask the endotoxin so that it will exhibit its full biological activity, e.g. pyrogenicity.
Body fluids are also known to contain inhibitors to the Limulus amoebocyte lysate test, Riegle and Cooperstock, Laboratory Medicine, 8, 28, (1977). There have been a number of attempts to inactivate Limulus amoebocyte lysate inhibitors in serum and plasma, Journal of Laboratory and Clinical Medicine, 75, 903 (1970); Proceedings of the Society of Experimental Biology and Medicine, 137, (1971); and Laboratory Medicine, 8, 28, (1977).
The above-mentioned difficulties are compounded by the extremely low concentrations of endotoxin in body fluids and the need to detect the endotoxins in the preshock stage of the infection. The invention overcomes the problems associated with detection and quantification of endotoxins in body fluids.